| Acute optic nerve injury caused by trauma, ischemia and glaucoma is one of the most important blind-induced diseases in ophthalmology. About 2-5 percent for cerebral trauma complication and there weren't ideal strategies for treatment. Optic nerve restoration research has become interesting and hot topic and it was also stringent for clinical treatment. There are two mainly obstacles that influence the optic nerve regeneration: One is the apoptosis,necrosis and autophagy of RGCs after nerve injury. RGCs were apoptosis through Caspase-3,RhoA/Rock, Nogo-A and P38 pathway[1-5] or lost the target-derived neurotrophic factor such as BDNF, CTNF and NGF [6,7]The other was known as critical obstacle that reactivity astrocyte respond to optic nerve injury to form glial scar barrier and secreted inhibitor extracellular matrix Chondroitin sulfate proteoglycan (CSPGs) to form chemical barrier.The methods to enhance axon regeneration had been reported previously. One is the target-derived neurotrophic factor like BDNF, CTNF and NGF supplemented exogenously,while the strategy rescued RGCs in temporary, overexpression of neurotrophic factor through trangesic method could maintain the RGCs survival just four weeks. Another methods is to disturb the apoptosis signaling pathway like caspase-3, Nogo-A and RhoA/Rock, the strategy could promote the cell survival in a degree while they couldn't enhance the axonal outgrowth and elongation[1,4,5].The last is to suppress the response of astrocyte to optic nerve injury and weak the glial scar barrier and inhibit microenvironment to promote the axon regeneration,while there were just a few axons regeneration and elongated a short distance[8-10].All above of the therauptic strategies got a short distance for axon regeneration and fail to functional restore. It suggests that there are multiple difficulties for axon regeneration after optic nerve injury and it is necessary to look forward a multi-target molecules for the treatment both through promoting the axon regeneration and suppressing the activation of astrocjytes.a crystallin is a major protein in the vertebrate lens and consist of two subunit A and B,which plays a crucial role in retinal ganglion cell sUrvival and axon regeneration [11-13].a crystallin acts as a therapeutic protein through its anti-apoptotic, anti-inflammatory and anti-aggregation [14]. It has reported previously that lens injury could stimulate axon regeneration in the optic nerve cut model and that the growth cone might reach the retinoreceptive layer of the superior colliculus at 5 weeks after optic nerve dlesion [15]. a crystallin promotes axon outgrowth by regulating the UroA/Rock signaling pathway[16],?crystallin also promotes rat retinal neurite growth on myelin substrates in vitro[17],promotes RGCs survival and inhibits microglial activation in vivo[18]. These study show aA crystallin might be a multi-target molecule. However, it remains unclear whether the axon could significantly penetrate the physical and chemical barrier following a crystallin treatment.BMP/Smad is an important signaling pathway for glial cell proliferation, activation and differentiation in central nerves system. After spinal cord injury, the expression of BMP2/4/7and phosphorylated Smadl/5/8 were promoted in oligodendrocytes and astrocytes[19]. Vivo study also observed neurosphere culture showed that BMP-4 promoted astrocyte differentiation from NSCs and suppressing production of neurons and oligodendrocytes.However, BMP4 blocked reagent Noggin notably decreased the ratio of astrocyte to neuron number[20],which suggest that BMP/Smad signaling take part in the proliferation,activation and differentiation of astrocytes. Another vitro study showed that the mRNA level of BMP and BMPR were promoted in optic nerve head astrocyte after ischemic-perfusion, additionally,the expression of BMP, BMPR and a crystallin protein also promoted[21], it is not clear whether the proteins could interact with each other. We previously observed both aA crystallin and aB crystallin could suppress the activation and proliferation of astrocytes in vitro, and the astrocyte cell scratch assay also showed that aA crystallin and aB crystallin,especially aA crystallin could inhibited astrocyte migration.Base on the reported description previously, one of the most important obstacles of axon regeneration after optic nerve crash is the reactivity astrocyte response to proliferation and migration to crush sit to form glial scar and inhibitory microenvironment. We hypothesize that aA crystallin enhanced axon regeneration and penetrated the crush site by suppressing the glia scar formation and decreasing the inhibitory molecular secretion,the mechanism of aA crystallin influence the astrocyte might be BMP/Smad signaling pathway. In present study, we established an optic nerve crush (ONC) rat model and aA crystallin (10-4g/l, 4?l) or PBS was intravitreous injected into the Sprague-Dawley rat model of ONC and glial fibrillary acidic protein (GFAP) expression in retinas and crush site was observed at 1 day, 3 days, 5 days, 7 days and 14 days after ONC through immunohistochemistry (IHC) and western blot (WB).Beta III tubulin (TUJ1), growth associated phosphoprotein-43 (GAP-43), chondroitin sulfate proteoglycan (CSPGs) and Neurocan were investigated at 14 days after ONC through IHC and WB. Flash visual evoked potential (F-VEP) was also detected in 14 days after ONC. In vitro, primary optic nerve astrocyte was cultured and isolated, scratch assay and oxygen-glucose deprivation (OGD) model would be used to observe the influcenec of aA crystallin to astrocyte migration and activation. Additonaly, the mRNA level of BMP/Smad signaling molecules also observed. Our study would provide a strategy to cure optic nerve injury diseases.Our study consists of three parts:Part one: The change of RGCs and glial activation in Sprague Dawley rats retinas and optic nerve after optic nerve crush.The SD rats' optic nerve was clamped at 2 mm behind the eyeball for 10 seconds using an artery clamp to cause moderate injury as previously reported. Just 3 days after optic nerve injury, we observed the RGCs decreased severely, the number of RGCs apoptosis was about 52% at 14 days after optic nerve crush. GFAP expression increased in the optic nerve at only 1 day after ONC and reached a peak level at 14 days (increased approximately 1.2-fold).Similarly, the GFAP levels of retinas increased significantly after the operation. In the optic nerve crush injury group, the astrocytes were degenerated and formed a GFAP-immunofluorescence reactivity free (GFAP-IR-free) zone after 3 days. The astrocytes at the distal side of the optic nerve migrated into this zone and formed glial scar, the arrangement of the astrocyte processes at the marginal crush site was arbitrary. Meanwhile,the waves of F-VEP were sharply decrease and became silent, the amplitude N1-P1 decreased to 5.24±1.67uV while the potency of P1 delayed to 126.75±8.94ms.Part two: aA crystallin intravitreal injection influence the astrocyte activation,TUJl-positive process surviual and axon regeneration at 14 days after optic nerve crushaA crystallin (10-4g/l; 4?l) (recombinant human CRYAA, Cell Signaling Technology,USA) was dissolved in sterile PBS in water, then injected into vitreous cavity using a 10?l volume microinjector, taking care not to injure the lens. The same volume (4?l) of sterile phosphate buffered saline(PBS )was injected in the PBS control group after optic nerve crush.We observed the GFAP expression and glial astrocyte morphology through IHC and WB found that aA crystallin suppressed the activation and morphological remodeling of astrocytes at the optic nerve crush site. Furthermore, the arrangement of astrocytes around the crush site was less arbitrary, the astrocyte migration into the GFAP-IR-free zone was inhibited, the molecular CSPGs and Neurocan also decreased after aA crystallin treatment. We observed the TUJ1 and GAP-43 expression and found that aA crystallin increased the number of TUJ1-positive process and enhanced axon regeneration and penetrated the crush site. F-VEP also restored obviously after aA crystallin treatment, the amplitude increased from 5.83±2.64?v (injury only group) to 12.02±1.64 ?v (aA crystallin treated group),the latency P1 was promoted from 126±9ms (injury only group) to 100±9ms (aA crystallin treated group).Part Three: the mechanism of a A crystallin suppress the proliferation and activation of astrocyte after oxygen-glucose deprivation (OGD) in vitro.Firstly, we cultured and isolated the primary optic nerve astrocyte in vitro to explore the mechanism of aA crystallin influence the astrocyte activation and glial scar formation, the purity of astrocyte was identified by GFAP positive expression while the GS or Ibal expression is negative. Secondly, we investigated the influence of a A crystallin in astrocyte migration through scratch assay. A final concentration 10-4g/l of aA crystallin was added in treatment group after scratch while the BSA (10-4g/l) was added in control group. We found the migration of astrocyte was delayed by aA crystallin treatment. The slower migration velocity was observed in aA crystallin treatment group.OGD was used in astrocyte activation in vitro. We divided normal culture group (control),OGD only group (OGD), OGD+BSA protein group (OGD+BSA) and OGD+aA crystallin treatment group (OGD+aA). We observed the GFAP and Neurocan expression by ICC and WB, and found OGD activated the astrocyte proliferation and secretion, GFAP and Neurocan expression level was increased, while after the aA crystallin treatment expression level was decreased. It is coincidence with our research in vivo. We also observed the mRNA expression level of BMP/Smad signaling molecules, we found BMP2/4 and Smadl/8 mRNA level was decreased after aA crystallin treatment. It suggest that aA crystallin might inhibit the activation, gliosis and secretion through BMP/Smad signaling pathway.Conclusion:1. Optic nerve crush induced the RGCs apoptosis as Caspase-3, RhoA/Rock, Nogo-A and P3 8 pathway activation or lost the target-derived neurotrophic factor. Astrocyte also activatied, proliferated and secreted inhibit moleculars to form glial scar and inhibitor environment prevented the RGCs axon regrowth and elongation. We observed aA crystallin treatment could promote the RGCs axon survival and restore F-VEP partially. Furtherly, aA crystallin could suppress the activation, migration, remodeling of astrocytes and inhibited the molecular CSPGs expression to enhance axon regeneration and penetrate the crush site. Our study shows aA crystallin is a multi-target molecule for ONC treatment and provide a strategy to cure optic nerve injury diseases.2. We observed that aA crystallin could suppress the activation, proliferation and inhibitory molecular expression of astrocyte through BMP/Smad pathway. |
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